Technology is rapidly advancing in the area of amplification and detection of nucleic acids, particularly as it relates to commercial diagnostic tests, which offer early detection of infectious diseases, cancer and genetic disorders. Highly sophisticated techniques for the amplification of minute quantities of nucleic acids, such as PCR (polymerase chain reaction), are now well known (see. U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188). The inherent sensitivity of PCR, i.e., its ability to amplify very small concentrations of a target DNA, means that low level carryover of PCR products, and contamination between specimens, can yield false positive results. Carryover and sample contamination, among other things, are a function of the number of manipulations of the sample required during processing. Therefore, a simple procedure with a minimal number of steps that expose the sample to the environment is highly desirable.
Traditionally, PCR identification of viremia due to infectious human cytomegalovirus (HCMV), and identification of other viruses and bacteria has been performed on peripheral blood leukocytes (WBCs) obtained from whole blood. Separation of WBCs from whole blood is usually required prior to extraction of the HCMV DNA from the WBCs. Procedures for this separation include erythrocyte sedimentation in a dextran solution (Rasmussen et al., J. Infect. Dis. 171:17714 82, 1995), differential lysis using ammonium chloride solutions (U.S. Pat. No. 5,702,884 Ekeze et al), or the use of commercially available procedures (such as, e.g., CPT-Vacutainer.TM. tubes from Becton-Dickinson). Typically, these procedures include a wash step which results in the removal of potential inhibitors of amplification; alternatively, the isolation of WBCs serves to remove these inhibitors, which typically reside in high concentration in plasma or serum.
Once the WBCs have been isolated, they are lysed and the DNA is extracted. This involves procedures such as, for example, boiling, sonication, or freeze-thawing of the WBCs, or the use of proteolytic enzymes and/or surfactants to lyse the cells and extract the DNA. DNA extraction may also involve an alkali lysis step (U.S. Pat. No. 5,639,599). Often, a more rigorous extraction/purification is performed to further ensure that purified DNA is obtained devoid of potential inhibitors, including, e.g., use of glass beads (Gene Clean II kits, Bio 101, Inc.), phenol-chloroform extraction procedures, polymer capture (U.S. Pat. No 5,582,988), spin-column adsorption (Qiagen QIAamp kits), and other commercially available DNA isolation kits (Puregene, Gentra Systems Inc.).
Notably, procedures used for isolating and lysing WBCs, which serve to wash or remove potential inhibitors from the WBC preparation, cannot be used with plasma and serum. As the vast majority of endogenous biochemical substances and consumed drugs, metabolites of drugs, and the like, reside and are heavily concentrated in serum and plasma, there is a need in the art for a rapid and robust procedure that could be used with serum and plasma which would remove the potential inhibitors or render them ineffective.
Extracellular HCMV nucleic acid in infected individuals is present in plasma and serum. Serum and plasma are gaining acceptance as samples of choice for detecting HCMV nucleic acid using PCR. Generally, the target DNA does not exist as free DNA but rather as a complex association of DNA, RNA, and proteins. The DNA must be extracted from the complex and denatured in order to render it available for amplification. Serum and plasma samples are typically subjected to heat, surfactants, and treated with proteases. Often additional rigorous protocols are employed to extract the target DNA, such as those described above for WBCs (Spector et al., J. Clin. Microbiol.30:2359-65, 1992; Nolte et al., J. Clin. Microbiol. 33:1263-66, 1995; Wolf et al., Transplantation 56:330-4, 1993; Patel et al., J. Clin. Microbiol. 32:1431-4, 1994). Alkali treatment has also been used. However, this alkali treatment typically requires a high NaOH concentration followed by a neutralization step (Hansen et al., J. Infect. Dis. 170:1271-4, 1994).
Thus, there is a need in the art for a rapid and efficient procedure for extracting DNA from serum and plasma that is compatible with PCR amplification methods.